1. Field of the Invention
This invention relates to a sample analysis apparatus and an analysis method. This invention particularly relates to a sample analysis apparatus, wherein a light beam is reflected from an interface between a thin film layer, which is in contact with a sample, and a dielectric material member, and an alteration occurring with an intensity of the reflected light beam is measured for an analysis of the sample. Also, this invention particularly relates to an analysis method, wherein an attenuated total reflection angle of the sample is measured by use of the sample analysis apparatus.
2. Description of the Related Art
As sample analysis apparatuses utilizing evanescent waves, surface plasmon sensors have heretofore been known. In metals, free electrons vibrate collectively, and a compression wave referred to as a plasma wave is thereby produced. The compression wave occurring on the metal surface and having been quantized is referred to as the surface plasmon. With the surface plasmon sensors, characteristics of samples are analyzed by the utilization of a phenomenon, in which the surface plasmon is excited by a light wave. Various types of surface plasmon sensors have heretofore been proposed. As one of well known surface plasmon sensors, a surface plasmon sensor utilizing a system referred to as the Kretschman arrangement may be mentioned. The surface plasmon sensor utilizing the system referred to as the Kretschman arrangement is described in, for example, Japanese Unexamined Patent Publication No. 6(1994)-167443.
Ordinarily, the surface plasmon sensor utilizing the system referred to as the Kretschman arrangement comprises (i) a dielectric material member having, for example, a prism-like shape, (ii) a metal film, which is formed on one surface of the dielectric material member and is brought into contact with a sample, (iii) a light beam irradiating optical system for producing a light beam and irradiating the light beam to an interface between the dielectric material member and the metal film at various different incidence angles such that a total reflection condition may be obtained at the interface between the dielectric material member and the metal film, and (iv) a detector for detecting the intensity of the light beam, which has been totally reflected from the interface described above.
With the surface plasmon sensor having the constitution described above, in cases where the light beam impinges at a specific incidence angle θSP, which is not smaller than the total reflection angle, upon the metal film, an evanescent wave having an electric field distribution occurs in the sample, which is in contact with the metal film, and the surface plasmon is excited by the evanescent wave and at the interface between the metal film and the sample. In cases where the wave vector of the evanescent wave coincides with the wave vector of the surface plasmon, and wave number matching is thus obtained, the evanescent wave and the surface plasmon resonate. (The thus occurring resonance is referred to as the surface plasmon resonance.) Energy of the light thus transfers to the surface plasmon. As a result, the intensity of the reflected light beam, which is totally reflected from the interface between the dielectric material member and the metal film, becomes markedly low. (The phenomenon, in which the intensity of the reflected light beam thus becomes markedly low, is referred to as the attenuated total reflection.) Ordinarily, the lowering of the intensity of the reflected light beam is detected as a dark line by the detector described above.
The surface plasmon resonance described above occurs only in cases where the incident light beam is a P-polarized light beam. Therefore, it is necessary for the incident light beam to be set previously so as to impinge upon the aforesaid interface as the P-polarized light beam.
The specific incidence angle θSP, which is associated with the lowering of the intensity of the reflected light beam, will hereinbelow be referred to as the attenuated total reflection angle (ATR angle) θSP. In cases where the wave number of the surface plasmon is found from the ATR angle θSP, a dielectric constant of the sample is capable of being calculated. Specifically, the formula shown below obtains.
            K      SP        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein KSP represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the light velocity in a vacuum, ∈m represents the dielectric constant of the metal, and ∈s represents the dielectric constant of the sample.
Specifically, in cases where the dielectric constant ∈s of the sample is found, the refractive index of the sample, or the like, is capable of being found in accordance with a predetermined calibration curve, or the like. Therefore, in cases where the ATR angle θSP is found, the dielectric constant ∈s of the sample is capable of being calculated. Accordingly, the characteristics with regard to the refractive index of the sample are capable of being calculated.
Besides the surface plasmon sensor, as a similar sensor utilizing the evanescent wave, a leaky mode sensor has heretofore been known. (The leaky mode sensor is described in, for example, “Surface Refracto-Sensor using Evanescent Waves: Principles and Instrumentations” by Takayuki Okamoto, Spectrum Researches, Vol. 47, No. 1, 1998.) Basically, the leaky mode sensor comprises (i) a dielectric material member having, for example, a prism-like shape, (ii) a cladding layer, which is formed on one surface of the dielectric material member, (iii) an optical waveguide layer, which is formed on the cladding layer and is brought into contact with a sample, (iv) a light beam irradiating optical system for producing a light beam and irradiating the light beam to an interface between the dielectric material member and the cladding layer at various different incidence angles such that a total reflection condition may be obtained at the interface between the dielectric material member and the cladding layer, and (v) a detector for detecting the intensity of the light beam, which has been totally reflected from the interface described above. The state of excitation of a guided mode is specified in accordance with the result of the detection having been made by the detector, and an analysis of the sample is thereby made.
With the leaky mode sensor having the constitution described above, in cases where the light beam impinges at an incidence angle, which is not smaller than the total reflection angle, upon the cladding layer via the dielectric material member, only the light having a certain specific wave number, which light has impinged at a specific incidence angle upon the cladding layer, is propagated in the guided mode in the optical waveguide layer after passing through the cladding layer. In cases where the guided mode is thus excited, approximately all of the incident light is taken into the optical waveguide layer, and the attenuated total reflection thus occurs. Also, the wave number of the guided optical wave depends upon the refractive index of the sample, which is located on the optical waveguide layer. Therefore, in cases where the ATR angle θSP is detected, the refractive index of the sample and characteristics of the sample with regard to the refractive index of the sample are capable of being analyzed.
In the fields of pharmaceutical research, and the like, the surface plasmon sensor and the leaky mode sensor described above are often utilized for random screening for finding out a specific substance, which is capable of undergoing the binding with a desired sensing substance. In such cases, the sensing substance is fixed to the aforesaid thin film layer (the metal film in the cases of the surface plasmon sensor, or the combination of the cladding layer and the optical waveguide layer in the cases of the leaky mode sensor), and a liquid (a liquid sample) containing a test body is introduced on the sensing substance. Also, at each of stages after the passage of predetermined periods of time, the aforesaid ATR angle θSP is measured.
In cases where the test body contained in the liquid sample is a substance capable of undergoing the binding with the sensing substance, the refractive index of the sensing substance alters with the passage of time. Therefore, the aforesaid ATR angle θSP is measured at each of stages after the passage of predetermined periods of time, and a judgment is made as to whether an alteration of the ATR angle θSP has been or has not been occurred. In this manner, a judgment is capable of being made as to whether the binding of the test body with the sensing substance has or has not occurred, i.e. as to whether the test body is or is not the specific substance capable of undergoing the binding with the sensing substance. Examples of the combinations of the specific substances and the sensing substances include the combination of an antigen and an antibody and the combination of an antibody and a different antibody. Specifically, examples of the analyses with regard to the combinations of the specific substances and the sensing substances include an analysis, wherein a rabbit anti-human IgG antibody is employed as the sensing substance, a detection is made as to whether a human IgG antibody acting as the test body has or has not been bound to the rabbit anti-human IgG antibody, and a quantitative analysis of the human IgG antibody is made.
In order for the state of the binding of the test body, which is contained in the liquid sample, with the sensing substance to be detected, the ATR angle θSP itself need not necessarily be detected. Alternatively, for example, the liquid sample containing the test body may be introduced onto the sensing substance, and thereafter the quantity of the alteration of the ATR angle θSP may be measured. Also, the state of the binding of the test body with the sensing substance may be detected in accordance with the quantity of the alteration of the ATR angle θSP.
As a technique for obtaining the various different incidence angles described above in each of the surface plasmon sensor and the leaky mode sensor, there has been known a technique, wherein a light beam having a small beam diameter is successively caused to impinge upon the aforesaid interface with the incidence angle being altered. (The aforesaid technique for obtaining the various different incidence angles is described in, for example, Japanese Unexamined Patent Publication No. 10(1998)-239233.) In such cases, the reflected light beam, which is reflected from the interface with its reflection angle altering in accordance with the alteration of the incidence angle of the incident light beam, may be detected with a small single-cell type of detector, which moves by being interlocked with the alteration of the reflection angle. Alternatively, the reflected light beam may be detected with a single-cell type of detector or a multi-cell type of detector, which extends in the direction of alteration of the reflection angle. In this manner, the incidence angle at the time at which the dark line is detected is capable of being specified as the attenuated total reflection angle.
As a different technique for obtaining the various different incidence angles described above in each of the surface plasmon sensor and the leaky mode sensor, there has been known a technique, wherein a light beam, which has a large beam diameter and is constituted of light beam components having various different incidence angles, is caused to impinge upon the aforesaid interface in a state of converged light or in a state of divergent light. (The aforesaid different technique for obtaining the various different incidence angles is described in, for example, Japanese Unexamined Patent Publication Nos. 6(1994)-167443 and U.S. Pat. No. 5,912,456.) In such cases, the light beam components, which have been reflected from the interface at various different reflection angles, may be detected with a multi-cell type of line sensor or a two-dimensional array sensor, which extends in a direction such that the sensor is capable of receiving all of the light beam components having been reflected from the interface at various different reflection angles. In this manner, the incidence angle of a light beam component corresponding to the position, at which the dark line has been detected, is capable of being specified as the attenuated total reflection angle.
However, with the technique, wherein the light beam having the small beam diameter is successively caused to impinge upon the aforesaid interface with the incidence angle being altered, the problems are encountered in that the constitution for altering the incidence angle is not capable of being kept simple, and in that quick processing is not capable of being performed. Also, in cases where the incidence angle is altered by use of a mechanical movement mechanism, the problems occur in that the detection accuracy is limited by an angle setting accuracy of the movement mechanism.
Also, with the technique, wherein the light beam, which has the large beam diameter and is constituted of the light beam components having various different incidence angles, is caused to impinge upon the aforesaid interface in the state of converged light or in the state of divergent light, the problems are encountered in that it is necessary for the detector whose cost is high, such as the multi-cell type of the line sensor or the two-dimensional array sensor, to be utilized, and in that calculation processing with complicated algorithms is required for the detection of the position of the dark line. Further, the problems occur in that the accuracy, with which the position of the dark line is detected, is limited due to adverse effects of unevenness of the interface and a spread width of the dark line. (particularly, in cases where a light source other than a laser beam is utilized).